Nucleosides and Nucleotides. 105. DNA Bending in d(A)4 - d(T)4 TRACTS Containing 3-Deazaadenine or 7-Deazaadenine Substituted for Adenine§,1

Abstract

Self-complementary decadeoxyribonucleotides containing 3-deazaadenine or 7-deazaadenine in place of one of the adenine moieties in a parent sequence, d(GAAAATTTTC)_n, have been synthesized. After phosphorylation at their 5′-ends, the decamers were ligated to form multimers, which were analyzed by polyacrylamide gel electrophoresis. The multimers of the decamer containing 3-deazaadenine (3), d(GAA3ATTTTC)_n, showed decreased bending compared with the multimers of d(GAAAATTTTC)_n, while the decamer containing 3-deazaadenine at a different position, d(GAAA3TTTTC)_n, did not show any degree of bending. Also, the properties of migration of the multimers containing 7-deazaadenine in the gel will be discussed.     

Synthesis of deoxyoligonucleotides containing 7-deazaadenine: recognition and cleavage by restriction endonuclease Bgl II and Sau 3AI (nucleosides and nucleotides Part 55).

Deoxydecanucleotides having a recognition sequence of Bgl II and Sau 3AI, and their 7-deazaadenine analogs were synthesized. The decanucleotides containing 7-deazaadenine in place of adenine were partially resistant to the hydrolysis by Sau 3AI and strongly resistant to that by Bgl II. A new hypothesis on the mode of recognition and cleavage of specific nucleotide sequences by Bgl II, recognizing one strand and cleaving the other strand, is presented.     

Phosphoramidites and oligonucleotides containing 7-deazapurines and pyrimidines carrying aminopropargyl side chains

The synthesis of phosphoramidites containing 7-deazaguanine, 7-deazaadenine, uracil and cytosine carrying aminopropargyl chains is described. The corresponding oligonucleotides are stabilized in duplexes thermally as well as against degradation by exonucleases.     

PHOSPHORAMIDITES AND OLIGONUCLEOTIDES CONTAINING 7-DEAZAPURINES AND PYRIMIDINES CARRYING AMINOPROPARGYL SIDE CHAINS

The synthesis of phosphoramidites containing 7-deazaguanine, 7-deazaadenine, uracil and cytosine carrying aminopropargyl chains is described. The corresponding oligonucleotides are stabilized in duplexes thermally as well as against degradation by exonucleases.     

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.     

Minor-groove-modified oligonucleotides: synthesis of decadeoxynucleotides containing hypoxanthine, N2-methylguanine and 3-deazaadenine, and their interactions with restriction endonucleases Bgl II, Sau, 3AI, and Mbo I (Nucleosides and Nucleotides Part 75).

Decadeoxynucleotides containing hypoxanthine, N2-methylguanine, 3-deazaadenine in the recognition sequences of restriction endonucleases Bgl II, Sau 3AI, and Mbo I were synthesized. These decanucleotides modified in the base moieties facing in to the minor groove were strongly resistant to hydrolysis by Bgl II and partially resistant to that of Sau 3AI and Mbo I. The decadeoxynucleotide containing 3-deazaadenine in place of adenine was bound to Bgl II strongly, whereas the nucleotides containing hypoxanthine and N2-methylguanine were bound less tightly.     

8-aza-7-deazaadenine and 7-deazaguanine: synthesis and properties of nucleosides and oligonucleotides with nucleobases linked at position-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2'-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C8-(2'-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2'-deoxyisoguanosine.     

2-Amino-7-deazaadenine forms stable base pairs with cytosine and thymine.

2-Amino-7-deazaadenine ((AD)A) was incorporated into oligodeoxynucleotides (ODN) and their base-pairing properties with natural nucleobases were investigated. In melting temperature (T(m)) experiments, the duplex containing an (AD)A/C base pair showed a high stability comparable to that containing (AD)A/T base pair. Destabilization of the duplex usually observed for existing degenerate bases was not observed. However, the incorporation efficiency of dCTP was only 1.8% for TTP in single-nucleotide insertion reactions using polymerase.     

Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P.

The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2'-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2'-hydroxyl that, when substituted with 7-deazaadenine or 2'-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2'-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.     

7-substituted 8-aza-7-deazapurines and 2,8-diaza-7-deaza-purines: synthesis of nucleosides and oligonucleotides

7-Substituted 8-aza- 7-deazaadenosines 1a-e were synthesized by Sonogashira cross coupling from the corresponding 7-iodo nucleoside in 36-79% yields. Starting from 7-bromo (or 7-iodo)-8-aza-7-deazaadenine, 2a,b were obtained by acid-catalyzed glycosylation followed by deprotection in 53 and 35% yields, repectively. Compounds 2b was applied to cross coupling reaction to give 2c-d in 34-95% yield. Compounds 2a and 4b were further transformed to the phosphoramidites 5 and 6b in 9 and 49% overall yields, which were incorporated into oligonucleotides.     

Studies on the Chemical Synthesis of Potential Antimetabolites. 371. Synthesis of 3-Deazaadenine Nucleosides Modified at the Sugar Moiety

Abstract

Several types of 3-deazaadenine pentofuranosides, represented by 9-(3-deoxy-β-D-glycero-pent-3-enofuranosyl)-3-deazaadenine (1), 9-(5-deoxy-β-Q-erythro-pent-4-enofuranosyl)-3-deazaadenine (2) and 9-β-D-xylo-furanosyl-3-deazaadenine (3), were prepared starting from 6-chloro-9-β-D-ribofuranosyl-3-deazaadenine (4).     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

7-deazapurin-2,6-diamine and 7-deazaguanine: synthesis and property of 7-substituted nucleosides and oligonucleotides

The synthesis of 7-substituted 7-deazaguanine and 7-deazaadenine ribonucleosides 1-2, the incorporation of 3a-d into oligonucleotides, and the stability of the corresponding duplexes and base discrimination are described. The pKa values of 3-4 are determined.     

Hoogsteen-Duplex DNA: Synthesis and Base Pairing of Oligodeoxynucleotides Containing 1-Deaza-2′-deoxyadenosine

Abstract

Solid-phase synthesis of oligonucleotides containing 1-deazaadenine was carried out employing phosphonate and phosphoramidite chemistry. Hoogsteen base pairing was established for the duplex d(c¹A₂₀)·d(T₂₀).     

8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTIES OF NUCLEOSIDES AND OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT POSITION-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N⁸-(2′-deoxy-ribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C⁸-(2′-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2′-deoxyisoguanosine.     

7-DEAZAPURIN-2,6-DIAMINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTY OF 7-SUBSTITUTED NUCLEOSIDES AND OLIGONUCLEOTIDES

The synthesis of 7-substituted 7-deazaguanine and 7-deazaadenine ribonucleosides 1–2, the incorporation of 3a–d into oligonucleotides, and the stability of the corresponding duplexes and base discrimination are described. The pK_a values of 3–4 are determined.     

Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.     

Unique hole-trapping property of the degenerate base,2-amino-7-deazaadenine

An artificial degenerate nucleobase, 2-amino-7-deazaadenine (1) showed remarkably high hole-trapping efficiency, similar to that of ^ZG and superior to that of the GGG triplet. The hole-trapping efficiency varied with the counterbase that base-pairs with 1.     

7-SUBSTITUTED 8-AZA-7-DEAZAPURINES AND 2,8-DIAZA-7-DEAZA-PURINES: SYNTHESIS OF NUCLEOSIDES AND OLIGONUCLEOTIDES

7-Substituted 8-aza-7-deazaadenosines 1a–e were synthesized by Sonogashira cross coupling from the corresponding 7-iodo nucleoside in 36–79% yields. Starting from 7-bromo (or 7-iodo)-8-aza-7-deazaadenine, 2a,b were obtained by acid-catalyzed glycosylation followed by deprotection in 53 and 35% yields, repectively. Compounds 2b was applied to cross coupling reaction to give 2c-d in 34–95% yield. Compounds 2a and 4b were further transformed to the phosphoramidites 5 and 6b in 9 and 49% overall yields, which were incorporated into oligonucleotides.     

Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

A set of 6 base-modified 2′-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse restriction endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by restriction cleavage.